1. Field of the Invention
The present invention relates to the preparation of heterodimeric, disulfide-linked .alpha./.beta. T cell receptors (TcR) in solution.
2. Background of the Related Art
The availability of soluble T cell receptors would allow the isolation and identification of natural and artificial ligands for therapeutic application, and the study of membrane-bound proteins. Proteins and antibodies to soluble T-cell receptors may be used in diagnosis and treatment of autoimmune diseases. For example, in a model immune system, experimental allergic encephalomyelitis, antibodies directed against TcRs inhibited and even reversed the development of the disease. In addition, soluble TcRs may be applicable to preventing rejection during organ transplantation and as therapy to prevent autoimmune diseases.
Structural and functional analysis of T cell receptor-ligand binding would also be greatly advanced by the availability of an intact, assembled TcR in soluble form. The "wild type" TcR processing pathway requires that the TcR .alpha. and .beta. chains associate with the CD3 components in the endoplasmic reticulum before they are transported to the T cell surface as reported by Clevers et al., Annu. Rev. Immunol., 6, 629 (1988); Alarcon et al., J. Biol. Chem., 263, 2953 (1988); Berkhout et al., J. Biol. Chem., 263, 8528 (1988); Koning et al., J. Immunol., 140, 3126 (1988); and Lippincott-Schwartz et al., Cell, 54, 209 (1988). Charged residues within the otherwise hydrophobic transmembrane region may be responsible for these associations, see Morley et al., J. Exp. Med., 168, 1971 (1988). Since the amino terminal variable and junctional regions completely encode T cell receptor antigen specificity and Major Histocompatibility Complex (MHC) restriction, which is the property of a T cell to respond to fragments of antigen complexed to self MHC molecules of other cells, a number of strategies which replace or delete the TcR transmembrane region have been attempted for the production of soluble TcR molecules.
Soluble TcR molecules made using chinese hamster ovary (CHO) cells are described by Lin et al., "Expression of T Cell Antigen Receptor Heterodimers in a Lipid-Linked Form", Science, 249, 677-679 (August 1990) and Davis et al., "TCR Recognition and Selection In Vivo", Cold Spring Harbor Symposia on Quantitative Biology, LIV, 119-128 (June 1989). Both of these articles describe the use of a GPI linkage approach to produce soluble TcR molecules. However, the soluble TcR molecules utilizing CHO transfectants, rather than using T-cell lymphomas of the present invention, produce soluble TcRs which are structurally and functionally different than those produced by the present invention. The following differences were observed:
1. When testing the CHO cells utilized by Lin et al. and Davis et al., the inventors herein observed that CHO cells fail to produce the clonotypic epitopes, which would indicate that a T-cell specific processing event is necessary for correct T-cell receptor pairing.
2. The molecular weight of the soluble T-cell receptors produced in accordance with Lin et al. and Davis et al. is significantly lower than the molecular weight of bound T-cell receptors and of the soluble T-cell receptor molecules of the present invention.
3. The soluble T-cell receptors reported by Lin et al. and Davis et al. fail to block the T-cell clone of origin. It may be that their molecule is incorrectly processed in CHO transfectants.
4. Due to the lack of of a clonotypic antibody to the 2B4 TcR, the 2B4 .alpha. and .beta. chains were shown to be associated by crossblocking experiments using anti-V.sub..alpha. and anti-V.sub..beta. specific monoclonal antibodies by FCM analysis. Thus it is impossible to judge if they are correctly paired.
Attempts to produce a soluble counterpart of the membrane bound T cell receptor by fusing TcR variable regions to immunoglobulin constant regions have produced, with one exception, unpaired .alpha. and .beta. chains, see Traunecker et al., Immunol. Today, 10, 29 (1989), and Gascoigne et al., Proc. Natl. Acad. Sci. USA, 84, 2936 (1987). These investigators claim to have produced soluble TcRs by techniques other than using the GPI linkage approach of the present invention. For example, Traunecker et al., Immunol. Today, 10, 29 (1989), Gascoigne et al., Proc. Natl. Acad. Sci. USA, 84, 2936 (1987) and Gascoigne, J. Biol. Chem., 265(16), 9296-9301 (June 1990) describe the use of myeloma cells J558L to produce and secrete a form of the TCR .beta. chain. Also see Gascoigne et al., Proc. Natl. Acad. Sci. USA, 88, 613-616 (January 1991) which describes the binding of SEA (staphyloccal entertoxin A) Raji cells, a Burkitt lymphoma cell that expresses MHC Class I and II, to piates coated with the soluble V.sub..beta..
Additionally, Mariuzza et al., J. Biol. Chem., 264(13), 7310-7316 (May 1989) utilize J558L myeloma cells to produce soluble .alpha. chains of the TcR. Traunecker et al., Eur. J. Immunol., 16, 851-854 (1986); and Becker et al., Cell, 58, 911-921 (September 1989) both use these same myeloma cells, however, neither report producing soluble TcR's.
Attempts to produce soluble .alpha./.beta. TcR molecules by the introduction of translational termination codons upstream of the TcR transmembrane region have also failed, as reported by Traunecker et al., Immunol. Today, 10, 29 (1989), and Gascoigne, J. Biol. Chem., 265, 9296 (1990). These methods include the production of TcRs/immunoglobulin chimeric proteins and the insertion of stop codons in front of the transmembrane portion of the TcR .alpha. and .beta. chains. These techniques utilized a stop codon to produce unpaired .alpha. or .beta. chains. Therefore, they produced a molecule which may not be functional, or is structurally quite different than that produced by the present invention. See Rebai et al., W-12 Structure/function relationships of the T cell receptor complex, (Abstract 12-11), 55 (1989) which reports production of soluble TcR .alpha. and .beta. chains using transfected myeloma cell lines. Their work, however, has never been published, and although their molecule is functional, their technique has failed to be broadly applicable to other TcR molecules; see Traunecker et al., (1989) supra. See also Gregoire et al., Proc. Natl. Acad. Sci., U.S.A., 88, 8077-8081, (September 1991) which reports that V.sub..alpha. C.sub..alpha. C.sub.k and V.sub..beta. C.sub..beta. C.sub.k were secreted as non covalent heterodimers and react with an anti-clonotypic antibody and two antibodies directed to the C domain of the TcR.
Guy et al., "Antigen-Specific Helper Function of Cell-Free T Cell Products Bearing TcR V.sub..beta. 8 Determinants", Science, 244, 1477-1480 (June 1989) report isolating TcRs from the supernatant of cloned T-helper cells. The molecular size of these TcRs was reported to be approximately 500 kD in its native state, which Guy et al. indicated would probably exist in a large complex. It, therefore, appears that these probably represent transmembrane TcRs in micelles. Therefore, Guy et al.'s TcRs are not truly soluble, and accordingly, are quite different than the present invention.
Goverman et al., Cell, 60, 929-939 (March 1990) report constructing polymeric receptor chains in which an immunoglobulin heavy chain variable region from a phosphorylcholine-specific antibody was substituted for a TcR .alpha. and .beta. variable regions. The modified receptor chain constructs were transfected into the mouse T-cell lymphoma EL4, and drug resistant clones were isolated. Goverman et al., however, utilize this construction to investigate the structure of antibodies rather than producing soluble TcRs.
In conclusion, none of the related art describe the production of soluble TcRs which have the same structure and molecular weight of the soluble TcRs of the present invention. In addition, the process for producing the soluble TcRs of the present invention is quite different than any of the techniques reported in these publications.